Healthcare workers (HCWs) are at a high risk for exposure to pathogens in the workplace. The objective of this study was to evaluate HCW adherence to follow-up after occupational exposure to blood and body fluids at a tertiary care university hospital in the city of São Paulo, Brazil. Data were collected from 2102 occupational exposures to blood and body fluids reports, obtained from the Infection Control Division of the Universidade Federal de São Paulo/Escola Paulista de Medicina/Hospital São Paulo, in São Paulo, Brazil, occurring between January of 2005 and December of 2011. To evaluate adherence to post-exposure follow-up among the affected HCWs, we took into consideration follow-up visits for serological testing. For HCWs exposed to materials from source patients infected with human immunodeficiency virus (HIV), hepatitis B virus (HBV), or hepatitis C virus (HCV), as well as from source patients of unknown serological status, follow-up serological testing was scheduled for 3 and 6 months after the accident. For those exposed to materials from source patients co-infected with HIV and HCV, follow-up evaluations were scheduled for 3, 6, and 12 months after the accident. During the study period, there were 2056 accidental exposures for which data regarding the serology of the source patient were available. Follow-up evaluation of the affected HCW was recommended in 612 (29.8%) of those incidents. After the implementation of a post-exposure protocol involving telephone calls and official letters mailed to the affected HCW, adherence to follow-up increased significantly, from 30.5 to 54.0% (P = 0.028). Adherence was correlated positively with being female (P = 0.009), with the source of the exposure being known (P = 0.026), with the source patient being HIV positive (P = 0.029), and with the HCW having no history of such accidents (P = 0.047). Adherence to the recommended serological testing was better at the evaluation scheduled for 3 months after the exposure (the initial evaluation) than at those scheduled for 6 and 12 months after the exposure (P = 0.004). During the study period, there was one confirmed case of HCW seroconversion to HCV positivity. The establishment of a protocol that involves the immediate supervisor of the affected HCWs, in the formal summoning of those HCWs is necessary in order to increase the rate of adherence to post-exposure follow-up.