The southern cattle fever tick, Rhipicephalus microplus Canestrini, is widely distributed in tropical and subtropical regions, causing huge economic losses to milk and meat production. Application of acaricides is the most widely used control strategy, but because of increasing evidence of resistance to acaricide and environmental concerns, novel strategies are needed. Use of entomopathogenic nematodes is a promising alternative for tick control. Six species of nematodes, Steinernema riobrave Cabanillas, Poinar, and Raulston (355 strain), S. carpocapsae (Weiser) (All strain), S. feltiae Filipjev (SN strain), Heterorhabditis bacteriophora Poinar (VS strain), H. indica Poinar, Karunakar, and David (HOM1 strain), and H. floridensis Nguyen, Gozel, Koppenhöfer, and Adams (K22 strain) were tested for pathogenicity against R. microplus (Deutch strain). Engorged female ticks were exposed to 1,250, 2,500, and 5,000 infective juvenile nematodes for 24, 48, and 72 hours in Petri dishes, and tick mortality over time, pre-oviposition period, egg mass weight, reproductive index, percentage inhibition of oviposition, and hatching percentage were evaluated. Dose-dependent mortality was recorded in Heterorhabditis species S. riobrave and S. carpocapsae which increased with exposure time. Minimum LC₅₀ (95% CL) values of 1999 (1947–2052) and 69 (63–75) infective juveniles per Petri dish were calculated for S. riobrave and H. floridensis, respectively, at 72 hours of exposure. Infective juvenile concentration and exposure time did not influence the pre-oviposition period. Differences in egg mass weight and reproductive index among the treatments and check group were observed with respect to concentration and exposure time. Reduction in hatchability was recorded only in ticks exposed to S. riobrave. Overall, H. floridensis was the most virulent species against engorged R. microplus females, adversely impacting most biological parameters. S. riobrave showed potential especially because it is native to South Texas and commercially produced. Future studies are warranted to evaluate the potential of this nematode species for biological control of the cattle fever tick under field conditions.