Background Post-exposure prophylaxis (PEP) using highly active antiretroviral therapy (HAART) significantly reduces the risk of HIV infection to the victims. Evidence based practice has brought dynamicity in HAART regimens necessitating continual updates on knowledge of health care providers (HCPs) regarding PEP. Therefore, this study aimed to assess contemporary knowledge of HCPs on PEP three years after the introduction of Tenofovir based HAART regimens as a default for PEP in Tanzania.Methods A health care facilities based cross-sectional study was conducted between April and June 2018 in Ilala municipal Dar es Salaam, Tanzania. A consecutive sampling method was deployed and 233 HCPs were enrolled. Data were collected using a self-administered questionnaire with closed-ended questions. The level of knowledge was graded using Bloom’s cut off point. Chi-square and multinomial logistic regression tests were used to check for association at significance level of p<0.05.Results A total of 13 health facilities were visited and 233 HCPs were interviewed among them 30.9%, 5.6%, 58.4%, and 5.2% were medical doctors, pharmaceutical personnel, nurses and laboratory technician respectively. The majority 72.1% (168) of the participants were female and the majority 47.2% (110) of the HCPs were aged ≥55 years. Diploma (43.8% (102)) education level constituted the largest proportion of the participants and the majority 50.2% (117) were nurses. About 70% of the respondents had low to moderate knowledge. The majority 87.6%(204) of the study participants were knowledgeable on dosage duration for PEP. However, only 41.2% (96) knew the recommended ARV regimen for PEP in Tanzania and 48.9% (114) knew how soon PEP should be initiated to prevent HIV acquisition. Certificate holders were more likely to have low knowledge on PEP compared to diploma and degree holders aOR (95% CI) 14(3.9 – 50.8), p=<0.01. The main source of PEP information to study participants was HIV treatment guideline (29%) followed by seminars (22%).Conclusion The majority of the assessed HCPs had low to moderate knowledge on PEP. The level of knowledge on PEP among HCPs increased with the level of education whereby those with a degree are more knowledgeable.