This study investigated autochthonous yeasts and their functions in the spontaneous fermentation of freshly crushed pineapple juice samples collected from two different areas of both Thailand and Australia. Hanseniaspora uvarum and Pichia guilliermondii were the main yeast species observed on the fruit skins of Thai samples, and also in the fresh juice and ferments of all samples from both countries. P. guilliermondii was consistently present as the dominant species during the early stage of the fermentation, whereas H. uvarum became more prevalent towards the end of the six-day fermentation period, with populations increasing from an initial level of ∼5log CFU/mL to ∼8log CFU/mL at the end of fermentation. The ethanol levels in samples from both regions of Thailand were maximal at 2 days of fermentation, reaching ∼1 to 2% (v/v) but then declined thereafter. In contrast, in the Australian samples ethanol levels continued to increase over the entire six-day fermentation period and reached ∼3 to 4% (v/v). A significant decrease in citric acid and increase in lactic acid levels were observed throughout the fermentation period in the samples from Thailand, but not in those from Australia where the different acid contents (and pH) were relatively stable. The other wine yeasts and, in particular, Saccharomyces yeasts, were not found in any of sampled fermentation systems that is apparently different from the other fruit juices. These findings suggested that the freshly crushed pineapple juice may possibly have some effects on the other autochthonous yeasts having important role in alcoholic fermentation.