Antimicrobial coatings can reduce the occurrence of medical device-related bacterial infections. Poly(2-(dimethylamino ethyl)methacrylate) (pDMAEMA) is one such polymer that is being researched in this regard. The aims of this study were to (1) elucidate pDMAEMA’s antimicrobial activity against a range of Gram-positive and Gram-negative bacteria and (2) to investigate its antimicrobial mode of action. The methods used include determination of minimum inhibitory concentration (MIC) values against various bacteria and the effect of pH and temperature on antimicrobial activity. The ability of pDMAEMA to permeabilise bacterial membranes was determined using the dyes 1-N-phenyl-naphthylamine and calcein-AM. Flow cytometry was used to investigate pDMAEMA’s capacity to be internalized by bacteria and to determine effects on bacterial cell cycling. pDMAEMA was bacteriostatic against Gram-negative bacteria with MIC values between 0.1−1 mg/mL. MIC values against Gram-positive bacteria were variable. pDMAEMA was active against Gram-positive bacteria around its pK
a and at lower pH values, while it was active against Gram-negative bacteria around its pK
a and at higher pH values. pDMAEMA inhibited bacterial growth by binding to the outside of the bacteria, permeabilizing the outer membrane and disrupting the cytoplasmic membrane. By incorporating pDMAEMA with erythromycin, it was found that the efficacy of the latter was increased against Gram-negative bacteria. Together, the results illustrate that pDMAEMA acts in a similar fashion to other cationic biocides.