Abstract <p>In this study, we present the synthesis and in vitro biological assessment of new substituted arylazopyrazoles and chromens that incorporate an isoindoline-1,3-dione group, aiming to identify potential drug candidates with enhanced antimicrobial and anticancer properties. Thus, the coupling of 3-[4-(1,3-dioxoisoindolin-2-yl)phenyl]-3-oxopropanenitrile with aryl diazonium salt resulted in the synthesis of acetohydrazonoyl cyanide derivatives. These derivatives, upon further reaction with hydrazine hydrate in ethanol, led to the formation of corresponding amino-arylazopyrazole derivatives. Additionally, the reaction of 3-[4-(1,3-dioxoisoindoline-2-yl)phenyl]-3-oxopropanenitrile with salicylaldehyde and naphthaldehyde led to the formation of 2-[4-(2-imino-2<i>H</i>-chromene-3-carbonyl)phenyl]isoindoline-1,3-dione and 2-[4-(3-imino-3<i>H</i>-benzo[<i>f</i>]chromene-2-carbonyl)phenyl]isoindoline-1,3-dione, respectively. We evaluated their efficacy against HCT-116 human colon carcinoma cells and various pathogenic microorganisms. The results demonstrate that these compounds have varying degrees of cytotoxicity against the carcinoma cells, with some showing comparable or superior efficacy to doxorubicin. Additionally, certain compounds exhibited significant antibacterial activity against both gram-negative and gram-positive bacteria and notable antifungal activity. The results show differing levels of cancer cell growth inhibition and notable antimicrobial effects, underscoring the potential of certain compounds for continued research in the development of anticancer and antimicrobial medications.</p>