β-Lapachone (β-lap, 1), an ortho-naphthoquinone natural product isolated from the lapacho tree (Tabebuia avellanedae) in many regions of South America, has received extensive attention due to various pharmacological activities, such as antitumor, anti-Trypanosoma cruzi, anti-Mycobacterium tuberculosis, antibacterial, and antimalarial activities. Related mechanisms of β-lap have been widely investigated for a full understanding of its therapeutic potentials. Numerous derivatives of β-lap have been reported with aims to generate new chemical entities, improve the corresponding biological potency, and overcome disadvantages of its physical and chemical properties and safety profiles. This review will give insight into the pharmacological mechanisms of β-lap and provide a comprehensive understanding of its structural modifications with regard to different therapeutic potentials. The available clinical trials related to β-lap and its derivatives are also summarized.