Although medicinal plants are used extensively in Lesotho to treat sexually transmitted infections (STIs), the antimicrobial efficacy of the majority of these plants has hitherto not been evaluated against pathogens implicated in STIs. The purpose of the current study was to investigate the antimicrobial activity, provide basic phytochemical composition of species not previously screened and examine the cytotoxicity of the medicinal plants used traditionally to treat STIs in Maseru, Lesotho. A total of 20 plant species were investigated for antimicrobial activity against three prevalent causative pathogens of STIs, as well as Candida albicans. The minimum inhibition concentration (MIC) micro-titer plate dilution assay was used to assess antimicrobial activity. In addition, plant species where no previous phytochemistry is known were qualitatively screened for their phytochemical constituents. Medicinal plants were also assessed for cytotoxicity using the brine shrimp lethality assay. Plant selection was based on information obtained from an ethnobotanical survey carried out by interviewing traditional practitioners in the Maseru District of Lesotho. Bulbine narcissifolia (roots) were found to be the most active (MIC value of 0.04 mg/ml for the organic extract and 0.03 mg/ml for the aqueous extract) against Neisseria gonorrhoeae ATCC 19424. Helichrysum caespititium was found to be the most broad-spectrum antimicrobially active plant species with MIC values of 0.02 mg/ml against Candida albicans ATCC 10231, 0.10 mg/ml against Gardnerella vaginalis ATCC 14018 and 0.06 mg/ml against Neisseria gonorrhoeae ATCC 19424. In general, the plant species that exhibited noteworthy antimicrobial activity against the test STI pathogens were also found to contain phytochemicals that are known to have antimicrobial effects. In addition, the cytotoxicological evaluation of the plants revealed that only three plant species [Agave americana (leaves), Berkheya setifera (leaves) and Parapodium costatum (roots)] were cytotoxic in the brine shrimp lethality assay when both the organic and aqueous extracts were tested. The results of this study support, to some extent, traditional medicinal use of the evaluated plants for the treatment of STIs.