Temozolomide (TMZ) is a methylating agent with promising antitumour activity against primary or secondary brain tumours. Through the generation of a reactive intermediate, TMZ interacts with DNA at different base site positions, generating a wide spectrum of methyl adducts represented mainly by N-methylpurines (70%) while, at a lesser extent, by N3-methyladenine (9%) and O6-methylguanine (5%). The antitumour activity of TMZ has been primarily attributed to O6- methylguanine, since tumour cell sensitivity inversely correlates with the levels of O6-alkylguanine DNA alkyltransferase and requires an intact mismatch repair system. Even though the pharmacokinetics properties, favourable toxicity profile and antitumour activity against a broad range of tumour types render TMZ an attractive agent in oncology, resistance to the methylating agent occurs relatively often and strongly affects the rate and durability of clinical response in cancer patients. Thus, different strategies aimed at counteracting resistance and increasing the efficacy of TMZ have been designed and for many of them investigation is still underway. Herein, we provide an update on the latest findings of preclinical and clinical studies on TMZ in combination with resistance or biological modulators and anticancer drugs with different mechanisms of action.