Aim: The aims of this study were to evaluate the surface morphology and surface roughness of restorative materials containing glass ionomer, analyze Streptococcus mutans biofilm formation on the surface of materials, and determine the correlation between surface roughness and biofilm. Materials and Methods: Four restorative materials: resin-modified glass ionomer; giomer; amalgomer; and glass carbomer were used and for each material, 6 mm in diameter and 2 mm in thickness disc-shaped specimens were prepared to evaluate the surface morphology (n = 3), surface roughness (n = 16), and biofilm (n = 20). Surface morphology was analyzed with a scanning electron microscope. Surface roughness was evaluated via an atomic force microscope. The biofilm was evaluated by counting the colony-forming units. Surface roughness measurements were evaluated using a one-way analysis of variance and Tukey HSD test. Biofilm parameters were analyzed using the Kruskal-Wallis H and Mann-Whitney U test. Pearson's correlation test was used to determine the correlation between surface roughness and biofilm. Results: While the highest roughness values were obtained for amalgomer and glass carbomer, the lowest roughness values belonged to giomer and resin-modified glass ionomer. Statistically significant differences in the number of adherent bacteria were observed between the materials only on day 1. No statistically significant correlation was determined between surface roughness and biofilm. Conclusions: The resin content and small filler particle size of material positively affect surface roughness. However, there is no direct relationship between surface roughness and biofilm.