Introduction: To protect patients and healthcare providers from healthcare-associated infections (HAIs), standard precautions have been promoted universally. Nurses are the forefront force inpatient care; therefore, evidence-based and up-to-date knowledge and practices can play a vital role in helping nurses apply standard precautions (SPs). Methods: The analytical cross-sectional study design was used. Through purposive sampling, the data were collected from 320 nurses, via, a self-administered questionnaire, from April to May 2017, from a public tertiary level hospital in Islamabad. The validity and reliability of each section were established. Mean and standard deviation were computed for continuous variables__ampersandsignrsquo; whereas; frequencies with percentages were calculated for categorical variables. The Multiple linear regression was applied for inferential statistics, maintaining 95% CI for important associations. Results: Out of 320 participants, the mean age of the participants was 33.04 __ampersandsignplusmn; 6.65 years. More than half of the study participants (66.88%) were females__ampersandsignrsquo; whereas 33.13% were male nurses. The mean knowledge and practice were 19.6 __ampersandsignplusmn;3.3 and 13.00__ampersandsignplusmn; 5.23, respectively, with a maximum score of 24 and 25. The results showed that the knowledge of the participants was better as compared to practices, though, in general knowledge they were reported as relatively deficient. Continuing training sessions on SPs revealed a significant 0.66 (95% C.I; 1.07, 3.67) relationship with the knowledge score in the final model. Practices were found to be better. Similarly, the Hep. B vaccinated nurses showed a positive impact of 1.49 (95% C.I; 0.07, 2.91) on the level of practices in MLR. Conclusion: The overall level of knowledge and practices among female nurses regarding standard precautions were inadequate. The study findings highlighted the need to continue intensive and in-service trainings sessions on SPs of infection control, using innovative approaches.