Antibacterial coatings can prevent and treat medical device‐associated infections. We examined the antibacterial properties of coatings assembled from poly‐l‐lysine (PLL) and hyaluronic acid (HA). PLL/HA films were fabricated using layer‐by‐layer assembly with three different PLL MWs, differentiated by number of repeat units, that is, 33, 91, and 407 (denoted by PLL30, PLL90, and PLL400). Films assembled with all three PLL MWs completely inhibited the growth of planktonic, gram‐positive Staphylococcus aureus and methicillin‐resistant S. aureus and gram‐negative Pseudomonas aeruginosa and Escherichia coli over a 24‐h exposure. All three film architectures also inhibited S. aureus attachment by ~60–70% compared to non‐film‐coated surfaces, likely attributed to significant film hydration and electrostatic repulsion due to HA. The true differences in antibacterial efficacy between different PLL MWs were observed upon repeated exposure of PLL/HA to S. aureus every 24 h. We found that PLL400 films lost the ability to inhibit planktonic S. aureus growth after one use while PLL30 and PLL90 films were effective over 4–5 and 9–13 repeated exposures, respectively. Our experiments indicated that differences in efficacy were related to low in‐film mobility of PLL400 and also agreed with dissolution timescales for PLL30 and PLL90 films. © 2019 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 107A: 1324–1339, 2019.