Neem seed oil (NSO) of Azadirachta indica (Meliaceae) contains more than 100 determined biologically active compounds, and many formulations deriving from them showed toxicity, antifeedancy and repellence against a number of arthropod pests. However, it is widely known that botanical products can differ in their chemical composition and bioactivity, as function of the production site and production process. We used high-performance thin layer chromatography (HPTLC) to investigate differences in chemical constituents of NSOs from three production sites. HPTLC analyses showed several differences in chemical abundance and diversity among NSOs, with special reference to limonoids. Furthermore, the three NSOs and their fractions of increasing polarities [i.e. ethyl acetate (EA) fraction and butanol (BU) fraction] were evaluated for larvicidal toxicity and field oviposition deterrence against the Asian tiger mosquito, Aedes albopictus, currently the most invasive mosquito worldwide. Results from bioactivity experiments showed good toxicity of NSOs and EA fractions against A. albopictus fourth instar larvae (with LC50 values ranging from 142.28 to 209.73 ppm), while little toxicity was exerted by BU fractions. A significant effect of the production site and dosage was also found and is probably linked to differences in abundance of constituents among samples, as highlighted by HPTLC analyses. NSOs and EAs were also able to deter A. albopictus oviposition in the field (effective repellence values ranging from 98.55 to 70.10%), while little effectiveness of BU fractions was found. Concerning ovideterrent activity, no difference due to the production site was found. This is the first report concerning larvicidal toxicity of NSO against A. albopictus and ovideterrence against Culicidae in the field. The chance to use chemicals from the NSO EA fraction seems promising, since they are effective at lower doses, if compared to synthetic products currently marketed, and could be an advantageous alternative to build newer and safer mosquito control tools.