Callus culture is the method for secondary metabolite production. Using elicitors can stimulate the production of secondary metabolites. The objective is to determine the elicitor effect on biomass and bioactive compounds in Piper betle L. var Nigra callus. The elicitor used copper sulfate (CuSO4) with concentrations of 0.5; 1.0; 2.5 mg/L, with six replications. The culture medium used Murashige and Skoog (MS) medium. The incubation time was eight weeks. Identification of bioactive compounds carried out using Gas Chromatography - Mass Spectrometry (GC-MS). The results showed that 0.5 mg/L CuSO4 produced the highest fresh weight (0.6711 g) and contained the bioactive compound salicylic acid hydrazide (21.23%). In general, these compounds act as antimicrobials.