Chili (Capsicum annuum) consumption is often suggested, and using functional food cultivars is the most effective strategy post COVID-19 pandemic. Controlling chili breeding activity is one of the most effective methods to produce new hybrid varieties. However, the general combining ability (GCA), specific combining ability (SCA), and heterotic effect of functional biochemicals (polyphenol content, antioxidant activities, and α-glucosidase inhibitory compounds) remain poorly known in C. annuum. This study aimed to estimate these parameters in C. annuum by using five different genotypes and their hybrid combinations based on growth characteristics, yield, yield components, and fruit functional biochemicals. The F1 and F1R progenies were obtained from crosses in a greenhouse with a full diallel mating design. Each parent used in this study had a GCA advantage for each characteristic. The hybrid combination of IPB074 × IPB005 and IPB435 × IPB367 displayed the best yield results. However, the results indicated the opposite regarding α-glucosidase inhibitory compounds. The heterotic effect of functional biochemicals was observed for traits related to genotypes, polyphenol content, antioxidant activity, α-glucosidase inhibitory compounds, and similar properties related to yield and yield components, indicating their use in hybrid chili production.