Potato is the fourth most consumed crop in the world. More than half of the crop is stored for three to nine months at cold temperatures (3-10 °C) for the fresh and seed market. One of the main causes of fresh potato waste in the retail supply chain is the processing of fungal and bacterial rots during storage. Dry rot is a fungal disease that mainly affects the potato crop during storage and is responsible for 1% of tuber losses in the UK. It is produced by Fusarium spp., such as Fusarium sambucinum and F. oxysporum , which can lead to the accumulation of mycotoxins in the potato tuber. Little is known about the impact of environmental factors on the accumulation of mycotoxins in potato tubers. Understanding the ecophysiology of these fungi is key to mitigating their occurrence under commercial storage conditions. Therefore, this work aimed to elucidate the effect of three different temperatures (5, 10, and 15 °C) and two different water activities (a w ; 0.97, 0.99) on the ecophysiology and mycotoxin accumulation of F. sambucinum and F. oxysporum in a potato-based semi-synthetic medium. The mycotoxin accumulation was then studied in vivo , in potato tubers cultivated under organic farming conditions, stored for 40 days at 8.5 °C. Results showed that higher temperatures and a w enhanced fungal growth, lag time, and mycotoxin accumulation in vitro . Growth rate was 2 and 3.6 times higher when the temperature increased from 5 to 10 and 15 °C, respectively. Six different mycotoxins (T-2, HT-2, diacetoxyscirpenol, 15-acetoxyscirpenol, neosolaniol, and beauvericin) were detected in vitro and in vivo . T-2 was the most abundant mycotoxin detected in vitro , observing 10 6 ng of T-2/g media after 21 days of incubation at 10 °C and 0.99 a w . Due to the long period of time that potato tubers spend in storage, the fluctuations of environmental factors, such as temperature and relative humidity, could promote the development of fungal rot, as well as mycotoxin accumulation. This could result in important food and economic losses for the potato market and a threat to food safety., Competing Interests: The authors declare no conflicts of interest.