One of the main pests of commercial rose crops in Colombia is the phytophagous mite Tetranychus urticae Koch. To manage this pest, synthetic chemicals have traditionally been used, some of which are well known to be potentially toxic to the environment and humans. Therefore, alternative strategies for pest management in greenhouse crops have been developed in recent years, including biological control with natural enemies such as parasitoids, predators and entomopathogenic microorganisms as well as chemical control using plant extracts. Such extracts have shown toxicity to insects, which has positioned them as a common alternative in programs of integrated pest management. The objective of this study was to evaluate the effect of an unfractionated ethanolic extract of Cnidoscolus aconitifolius leaves on adult females of T. urticae under laboratory conditions. The extract was chemically characterized by recording its metabolic profile via liquid chromatography coupled to mass spectrometry, along with tentative metabolite identification. The immersion technique and direct application to rose leaves were used to evaluate the effects of seven doses (10–2,000 µg/mL) of the ethanol extract of C. aconitifolius leaves on T. urticae females under laboratory conditions. The mortality and oviposition of individuals were recorded at 24, 48 and 72 h. It was found that the C. aconitifolius leaf extract reduced fertility and increased mortality in a dose-dependent manner. The main metabolites identified included flavonoid- and sesquiterpene-type compounds, in addition to chromone- and xanthone-type compounds as minor constituents with potential acaricidal effects.