In vitro effect of 1% theobromine addition on the physical and chemical properties of conventional glass ionomer (GIC) cement was investigated. Conventional GIC (GIC-C) and 1% theobromine added to GIC (GIC-THEO) specimens were compared regarding the microhardness (n = 10), sorption (n = 5), solubility (n = 5), color change (n = 10), fluoride release in saliva (n = 10) and the amount of biofilm deposition (n = 20). Compared against conventional GIC, adding 1% theobromine increased microhardness (p < 0.05), while its sorption, solubility, color and fluoride release to saliva (p > 0.05) remained unchanged. On the other hand, Streptococcus mutans biofilm amount deposited on its surface decreased statistically when theobromine was added to GIC (p < 0.05). Based on the results, it could be concluded that 1% theobromine addition to GIC can be a good strategy as it keeps some of its properties and improves microhardness and biofilm deposits strengthening its role in the preventive approach of dentistry.