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We assessed the effect of daily cotrimoxazole, essential for HIV care, on development of antifolate-resistant Plasmodium falciparum, naso-pharyngeal Streptococcus pneumoniae (pneumococcus), and commensal Escherichia coli. HIV-positive subjects with CD4 cell count < 350 cells/μL (lower-CD4; N = 692) received cotrimoxazole; HIV-positive with CD4 cell count ≥ 350 cells/μL (higher-CD4; N = 336) and HIV-negative subjects (N = 132) received multivitamins. Specimens were collected at baseline, 2 weeks, monthly, and at sick visits during 6 months of follow-up to compare changes in resistance, with higher-CD4 as referent. P. falciparum parasitemia incidence density was 16 and 156/100 person-years in lower-CD4 and higher-CD4, respectively (adjusted rate ratio [ARR] = 0.11; 95% confidence interval [CI] = 0.06–0.15; P < 0.001) and 97/100 person-years in HIV-negative subjects (ARR = 0.62; 95% CI = 0.44–0.86; P = 005). Incidence density of triple and quintuple dihydrofolate-reductase/dihydropteroate-synthetase mutations was 90% reduced in lower-CD4 compared with referent. Overall, cotrimoxazole non-susceptibility was high among isolated pneumococcus (92%) and E. coli (76%) and increased significantly in lower-CD4 subjects by Week 2 (P < 0.005). Daily cotrimoxazole prevented malaria and reduced incidence of antifolate-resistant P. falciparum but contributed to increased pneumococcus and commensal Escherichia coli resistance.