Background: The study aim was to assess knowledge, and adherence of Health care workers (HCWs) to standard precautions among health care providers in Beni-Suef university hospital. Methods: A cross-sectional analytical study was conducted in Beni-Suef University Hospital who agreed to participate in the study (n=428). A self-administered structured questionnaire was prepared in Arabic language was distributed among participants. Results: A total of 428 health care workers from Beni-Suef University participated in the present study with a mean age of (29.51± 4.91). The mean of knowledge score of the standard precautions knowledge questionnaire (SPKQ) was (14.29 ±2.88) and mean of adherence score of the standard precautions adherence questionnaire (SPAQ) is (54.69 ±11.09). There was a significant moderate positive correlation between standard precautions knowledge and adherence with correlation coefficient (r) 0.303. Conclusion: The knowledge and adherence of HCWs of Beni-Suef university hospital towards standard precautions is low with a knowledge score of 14.29 out of 20 and adherence score was 54.69 out of 80. So strict observation of HCWs during work and through continuous evaluation of their adherence to SP and correction of their poor practices by the infection control team is required.