Aim: The study aimed to investigate the antifungal effects of herbal extracts and fluconazole on heat-polymerized acrylic denture base resin as a denture cleanser. Materials and methods: Several essential oils, such as origanum oil and grape seed oil and commercially available antifungal agent fluconazole were used as denture cleansers and their antifungal efficacy was evaluated using a spectrophotometer. Overall, 68 samples were obtained and were divided into four groups, each containing 17 samples. These samples were immersed in Sabouraud dextrose broth consisting of Candida albicans for 16 hours and later in these antifungal solutions for 8 hours and their antifungal efficacy was measured. Data were subjected to an ANOVA test. Results: Among the study groups origanum oil showed the maximum antifungal activity with a mean optical density at 0.072 ± 0.014 followed by fluconazole (0.094 ± 0.155), and least by grape seed oil (0.190 ± 0.071). Conclusion: Results of this in vitro study showed that origanum oil was more effective than commercially available antifungal agents, and among the tested groups oregano oil was a potential agent in lowering the C. albicans colony. Clinical significance: Origanum oil, being a herbal product, can be considered as a denture cleanser and also be used as an effective alternative to commercially available antifungal agents without any side effects.