Robert Mark Lightfoot, son of Archdeacon Thomas Fothergill Lightfoot, grew up in Cape Town and received his schooling at the Diocesan College. Here he met Louis A. Peringuey*, who taught French there, and shared his interest in natural history. In June 1882 the two found a rare tiger beetle, Platychile pallida, and presented it to R. Trimen* at the South African Museum. From then on Lightfoot regularly contributed specimens to the museum. On 1 April 1888 he joined its staff as an assistant, a position he retained for the rest of his life. His duties were varied, including those of clerk, bookkeeper, librarian, collection manager of molluscs, assistant to Peringuey, and collector. He was a capable naturalist and despite suffering from kyphosis (humpback) was an active collector of molluscs, insects, spiders and scorpions, mainly in the western Cape, both as part of his museum duties and in his private capacity.
Lightfoot's first entomological collecting trip was to Namaqualand in 1890 and resulted in a valuable collection of insects, including 30 species of Diptera and Hymeoptera new to the museum, of which several were new to science. In 1895 he found a new species of Peripatus, only the third species known in South Africa, on Lion's Hill, Cape Town. From about this time he collected marine invertebrates for W.F. Purcell*, until these activities were taken over by K.H. Barnard* around 1911. In 1897 he was in charge of the museum's mollusc collection and arranged specimens for display. During 1898 he helped the Director, W.L. Sclater* to completely reorganise the library and arrange it according to a modification of the Dewey system. That year he also collected some small mammals and lizards. The next year he went to Europe on holiday from April to October, but still found time to collect spiders at Clanwilliam, including 23 species new to the museum, and donated a small collection of molluscs, including eight species not yet represented in the collections. During the next few years he contributed arachnids, small moths and Tenebrionidae beetles, and in 1906 collected over 400 insects at Saldanha Bay with G.H. Herman*. In 1907 he served as the museum's librarian, but also collected plants at Houw Hoek with E.P. Phillips* and insects in the Caledon and Tulbach districts, securing valuable specimens. The next year, with J.M. Bain*, he investigated some caves that contained human remains. In 1910 he, E.P. Phillips and James Drury* collected insects, plants and human skeletons in Namaqualand. From 1909 to 1912 he donated various species of Stapelia, which he cultivated himself. Two of the species were new to the museum's herbarium.
During his long association with the museum Lightfoot contributed an enormous number of specimens to various departments. However, his speciality was shells, which he collected in Namaqualand, Namibia, the mountains of the Cape Colony, and at Beira. Several shell species were named after him, namely the marine species Terebra lightfooti and Columbella lightfooti, and the non-marine species Sheldonia lightfooti and Trachycystis lightfootiana. He is also commemorated in Lightfoot's Moss Frog, Arthroleptella lightfooti, named by G.A. Boulenger* in 1910. About 1907 he was elected a Fellow of the (British) Entomological Society. That year he became a member of the South African Philosophical Society and remained a member for a few years after it became the Royal Society of South Africa in 1908.
On 3 Aprlil 1913 Lightfoof married Gertrude Annie Lonsdale.