Objectives: A prospective survey was conducted to determine the prevalence of asymptomatic nasopharyngeal carriage of Streptococcus pneumoniae and Haemophilus influenzae type b in children under 5 years of age in Botswana and to determine the antibiotic resistance patterns of these organisms to commonly used antimicrobial agents. Methods: Children 2 months to 5 years of age (n = 249) were recruited from outpatient clinics in Gaborone and Francistown, and 29 were sampled from the pediatric wards at Princess Marina (Gaborone) and Nyangabgwe (Francistown) Hospitals. Nasopharyngeal specimens were collected and the carriage and antibiotic resistance of S. pneumoniae and H. influenzae type b were determined. Analyses of risk factors associated with carriage and resistance were performed. Results: Streptococcus pneumoniae was isolated from 69% of the outpatient children in Gaborone and 85% of the children in Francistown; the carriage rate in hospitalized children was 36% and 33% in Gaborone and Francistown, respectively. Approximately half of the isolates at both sites were resistant to at least one antibiotic, the most common being cotrimoxazole and penicillin. Resistance to three or more antibiotics (multiple resistance) was found in less than 10% of the isolates. Most penicillin resistance at both sites was at the intermediate level; however, almost 20% of the isolates demonstrated high-level resistance to cotrimoxazole. The most prevalent serogroups or serotypes of antibiotic-resistant isolates were 14, 19F, 19A, 6A, 6B, and 4. No risk factors for antibiotic resistance were identified. Haemophilus influenzae type b was isolated from 8% of the children in Gaborone and from 3% of the children in Francistown. Almost a third of the isolates were resistant to ampicillin. Conclusions: The high levels of antibiotic resistance in pneumococci isolated from children in Botswana suggest that the clinical management of meningitis and ottis media with a β-lactam antibiotic may fail in a significant proportion of cases and that empiric first-line use of cefotaxime or ceftriaxone for meningitis and higher dose amoxicillin (90 mg/kg/day) for otitis media is recommended. The levels of penicillin resistance in this study would not impact on the management of pneumonia with amoxicillin.