Hospital-acquired infections (HAIs) contribute to increased length of hospital stay, higher mortality and higher health-care costs. Prevention and control of HAIs is a critical public health concern. This study assessed the knowledge, attitude, and practice (KAP) of standard infection control precautions among health-care workers (HCWs) in Qassim, Saudi Arabia. A cross-sectional online survey among HCWs was conducted using a structured questionnaire. Predictors of KAP were investigated using multivariate logistic regression analyses and independent sample t-tests. A total of 213 HCWs participated in the survey. The prevalence of good (>80% correct response) knowledge, attitude, and practice were 67.6%, 61.5%, and 73.2%, respectively. The predictors of good knowledge included the age of the HCWs (>34 years) (adjusted odds ratio: 30.5, p < 0.001), and training (13.3, p < 0.001). More than 6 years of work experience was a significant predictor of having a positive attitude (5.5, p < 0.001). While the predictors of good practice were having >6 years of experience (2.9, p < 0.01), previous exposure to HAIs (2.5, p < 0.05), and training (3.5, p < 0.01). However, being female (0.22, p < 0.001) and older (>34 years) (0.34,p < 0.01) were negatively associated with knowledge. Results indicate that arranging training for HCWs might be useful in improving their knowledge of standard infection control precautions and is also expected to facilitate positive attitude and practice.