Purpose: To follow up the variations of early morning concentrations of oral volatile sulfur compounds (VSCs) in periodontally healthy subjects who refrained from toothbrushing during a period of 5 days while continuing with other hygiene procedures. Materials and Methods: Thirteen subjects volunteered for this study. At baseline, all subjects stopped brushing their teeth for 5 days, but continued using dental floss and a tongue scraper twice daily. Organoleptic measurements, VSC quantification and plaque index were recorded in all subjects at baseline and on days 3 and 5 of the study period. Results: At baseline, the visible plaque index was significantly lower than that observed on days 3 and 5 (P = 0.0001). However, no significant differences in the amount of plaque were observed between days 3 and 5 (P > 0.05). The results of the organoleptic measurements and VSC quantifications did not show any statistically significant differences between baseline and day 3, or between days 3 and 5 (P > 0.05). However, a significant increase was observed in both VSCs (P = 0.008) and organoleptic scores (P = 0.032) when baseline values were compared to day 5. A positive correlation between VSCs and organoleptic scores was observed in all measurements (r = 0.67, P 0.0001). Conclusion: The results of this study suggest that the lack of toothbrushing may affect VSC levels and the organoleptic perception of oral malodour due to the development of biofilm on buccal and lingual surfaces of teeth in healthy subjects. Keywords: morning bad breath, periodontally healthy subjects, toothbrushing, volatile sulfur compounds