Thirty nine ethanol extracts from 26 plants widely distributed in Botswana were screened for antimicrobial activities against three Grampositive, two Gram-negative bacterial strains, two Candida species and one Mycobacterium species. Screening was carried out at an initial concentration of 500 μg/disc using the disc agar diffusion method. Most of the plant species gave rise to antifungal activities (20/24), some of them specifically, such as Acrotome inflata, Bridelia mollis, Dichrostachys cinerea, Dicerocaryum eriocarpum, Dicoma capensis, Gomphrena celosioides, Tagetes minuta and Waltheria indica. Minimal inhibitory concentrations (MICs) of extracts with inhibitory activities against Candida albicans, a major opportunistic infective agent in immunocompromised patients, varied between 0.039 and 2.5 mg/ml. Extracts of 10 plant species inhibited the growth of Mycobacterium aurum, a non-pathogenic model organism with similar drug susceptibility as Mycobacterium tuberculosis. MIC values of anti-mycobacterial extracts ranged from 0.039 to 2.5 mg/ml. Interestingly, Ocimum canum leaf extracts and Elephantorrhiza burkei root extracts displayed the lowest MICs against both HIV/AIDS opportunistic pathogens with values of 0.039 mg/ml against Mycobacterium aurum, and MIC values of 0.039 and 0.078 mg/ml against Candida albicans, respectively. Extracts of two plant species, Elephantorrhiza elephantina and Persiceria limbata, exhibited antimicrobial properties against all eight microorganisms tested and only these two extracts were active against the Gram-negative Escherichia coli strain. Antimicrobial ethanol extracts with the lowest MIC values did not show acute in-vitro cytotoxicity up to a concentration of 1000 μg/ml using human embryonic kidney cells (HEK293). The findings confirm some anti-infective and wound healing ethnomedical uses in Botswana and show the potential to develop antimicrobial preparations from community natural resources.