Tackling the worldwide problem of antibiotic resistance requires addressing the lack of knowledge about antibiotics and understanding the impact of resistance. This study aimed to assess healthcare professionals' (HCP) knowledge about antibiotics and antibiotic resistance and the antibiotic prescription practices for outpatients at the Manhiça District Hospital in Mozambique. This cross-sectional study assessed the knowledge and practices of 20 HCPs about antibiotic use and resistance and evaluated quality indicators of antibiotic use of 200 prescriptions. We observed that 20% of the participants incorrectly placed amoxicillin in the cephalosporin group, and 10% considered antibiotic resistance a rejection reaction by the patient's body. However, the HCPs showed Knowledge level A. Antibiotics featured in 88% of prescriptions, with an average of one antibiotic per prescription. Cotrimoxazole (30.77%) and amoxicillin (26.15%) were the most frequently prescribed antibiotics. Cotrimoxazole was more prescribed for adults (21.54%) and amoxicillin for children (17.95%). Seasonal variation was observed with an increased winter consumption of cotrimoxazole and phenoxymethylpenicillin in summer. The results revealed a high level of knowledge of HCPs about how to use and identify antibiotics. A higher frequency of broad-spectrum antibiotic prescriptions was observed with cotrimoxazole and amoxicillin being the most prescribed.