The chemical composition and biological activity of a sample of yellow propolis from Mato Grosso do Sul, Brazil (EEP‐Y MS), were investigated for the first time and compared with green, brown, and red types of Brazilian propolis and with a sample of yellow propolis from Cuba. Overall, EEP‐Y MS had different qualitative chemical profiles, as well as different cytotoxic and antimicrobial activities when compared to the other types of propolis assessed in this study and it is a different chemotype of Brazilian propolis. Absence of phenolic compounds and the presence of mixtures of aliphatic compounds in yellow propolis were determined by analysing ¹H‐NMR spectra and fifteen terpenes were identified by GC‐MS. EEP‐Y MS showed cytotoxic activity against human tumour strain OVCAR‐8 but was not active against Gram‐negative or Gram‐positive bacteria. Our results confirm the difficulty of establishing a uniform quality standard for propolis from diverse geographical origins. The most appropriate pharmacological applications of yellow types of propolis must be further investigated.