The aim of this study was to investigate acid resistance induced by starvation or biofilm formation in Streptococcus mutans ATCC 25175. The artificial biofilms were made on cover glasses, starved for 24 h and immersed in 0.1 M lactate buffer at pH 3.8 for 10 min. The biofilms were also exposed to 5% sucrose solution for 20 min to simulate acid shock produced by sucrose metabolism. Confocal laser scanning microscopy with fluorescein isothiocyanate staining measured the resultant minimum pH in biofilms. Live and dead organisms in biofilms were differentiated by confocal laser scanning microscopy with proidium iodide and SYTO9 staining. The same processes were used to treat planktonic organisms. The results showed that starved biofilms or planktonic cells showed significantly more viable bacteria after acid shock induced either by lactic acid or during sucrose consumption than non-starved biofilms or planktonic cells. In addition, biofilms showed greater resistance to acid shock induced by lactic acid than planktonic cells, whereas similar results were obtained where sucrose was used as a carbon source to reduce pH in biofilms and planktonic cells. Thus, it is suggested that starvation protects both biofilm and planktonic S. mutans from acid shock induced either by lactic acid or during sucrose consumption, while biofilm formation seemed to protect bacteria only from acid shock induced by pH 3.8 lactate buffer but not the acid shock of a slightly higher pH produced during sucrose consumption.