Purpose: To compare surface roughness and bacterial colonization of Streptococcus mutans to 3D printed (3DP), milled (M), and conventional (CV) acrylic resin. Methods: Thirty-six discs (n equals 12 per group) were fabricated from 3DP, M, and CV materials. One surface of sample was polished (Po); the opposite surface was left unpolished (UPo). Surface roughness (μm) was assessed using a contact profilometer. The specimens were placed in S. mutans suspension and incubated at 37 degrees Celsius overnight. The attached colonies were separated using a sonicator, and the resulting solution was diluted to 10-3 to assess colony-forming units per milliliter (CFU/ml) after 48 hours. The colonies were categorized into a quantitative S. mutans (QS) index. Data were analyzed using one-way ANOVA, chi-squares, and multivariate analysis of variance analysis with the least significant difference (LSD) post-hoc test (P<0.05). Results: Roughness average (Ra) values of CV were higher than 3DP and M for UPo surfaces (P<0.001; 3DP=0.10; M=0.13; CV=0.26 μm, respectively). For Po and UPo surfaces, the CV harbored more S. mutans colonies than M and 3DP (P<0.001; 3DP=5.2x10 6 ; M=4.7x10 6 ; CV=1.49x10 7 CFU/ml, respectively). M group had the lowest range of QS scores, while CV had the highest range (P<0.001). Conclusions: Digitally manufactured material provides smoother surfaces than the conventional group, resulting in fewer Streptococcus mutans colonies. However, all the material groups must still be adequately polished to prevent the colonization of S. mutans, regardless of the manufacturing methods, as higher S. mutans counts were observed with an increase in surface roughness values.