AIMS: This article describes the qualitative and quantitative analyses of untypical compounds in the cuticular and internal lipids of four dipteran species. For isolated compounds, antimicrobial activity against 18 reference strains of bacteria and fungi was determined. METHODS AND RESULTS: In this study, gas chromatography (GC) combined with mass spectrometry (GC–MS) was used to analyse the surface and internal compounds of four fly species. Seven untypical compounds from both pre‐imaginal and imaginal stages of examined insects were identified. Azelaic acid (AA) was the most abundant, while phenylacetic and phenylpropionic acids occurred in lower concentration. Minor quantities of sebacic acid, 2‐methyl‐2‐hydroxybutanoic acid, tocopherol acetate and trace amounts of 2,4‐decadienal were also detected. Tocopherol acetate was found only in cuticular lipids of Musca domestica larvae. Each compound was tested against several species of fungi and bacteria by determining minimal inhibitory concentration (MIC). Human pathogenic fungi were also investigated. Phenylpropionic acid showed the greatest antifungal activity. Bacterial strains were insensitive to the presence of identified compounds, apart from 2,4‐decadienal which strongly inhibited bacterial growth. CONCLUSIONS: This is the first time that the chemical composition and the antimicrobial activity of untypical compounds in the cuticular and internal lipids of four fly species has been analysed. SIGNIFICANCE AND IMPACT OF THE STUDY: Determination of untypical compounds and their antimicrobial activity can effectively contribute to the knowledge concerning insect defence mechanisms.