Brazil is the first country in the world to have broad coverage standard (NR-32) focused on protecting health workers exposed to biological risks. This study evaluated the degree of knowledge of the NR-32 Standard and the level of knowledge and compliance with the standard precautions. A cross-sectional study was conducted with 208 randomly selected health professionals; 93 of them were residents and 115 were physicians at a Brazilian Clinical Hospital. To collect information, the participants were interviewed and/or they completed semi-structured questionnaires divided into three domains: knowledge of the standard, knowledge of biosafety, and compliance with standard precautions. Cronbach's alpha was used to assess internal consistency of the scales of knowledge and compliance with values above +0.75 indicating excellent agreement. Multivariate linear regression was used to evaluate the predictors for compliance with NR-32, biosafety, and standard precautions. Mean knowledge of the NR-32 Standard was 2.2 (± 2.02) points (minimum 0 and maximum 7 points). The minimum expected mean was 5.25 points. The mean knowledge of biosafety was 12.31 (± 2.10) points (minimum 4 and maximum 16 points). The minimum expected mean was 12.75 points. The mean compliance with standard precautions was 12.79 (± 2.6) points (minimum 6 and maximum 18 points). The minimum expected mean was 13.5 points. The individual means for using gloves, masks and goggles during procedures and for not recapping needles were 2.69, 2.27, 1.20 and 2.14, respectively. The factors associated with knowledge of the NR-32 were: greater knowledge amongst those who studied at a public university and who had knowledge of biosafety. The knowledge of the NR-32 Standard was low, but there was a good level of knowledge of biosafety issues. The compliance with standard precautions was acceptable in general, but was low for some of the evaluated precautions.