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 (1)H-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.