Germany's Verdi services union said the company was getting rich by "saving money on the health of its workers". Shoppers were expected to spend $3.4 billion on Amazon during the promotion, up more than 40 percent from last year's Prime Day, according to Coresight Research.
Amazon doesn't release Prime Day sales figures.
The Spanish Workers' Commissions (CCOO) and General Workers' Union (UGT) said almost 80 percent of workers left their positions at Amazon's largest packing and distributing centre in the country.
Following the launch of Prime in Australia last month, Amazon's app went from the ninth to the sixth most downloaded shopping app in the country.
Monday also saw Amazon's market capitalisation rise to $888bn, making it the world's second most valuable company after tech giant Apple, valued at $940bn.
Amazon's annual 36-hour shopathon, an important marketing tool as well as a boost to business, had been expected to drum up $3.4 billion of spending - up more than 40 percent from previous year - as bargain-hunters chase promotions and discounts.
"When traffic comes in such high volumes, it makes it hard to run the security to distinguish between a regular customer and a bot", Zilberman said.
Amazon's own online shopping holiday - Prime Day - was off to a rocky start when some shoppers found problems using the website when the hyped-up sales were supposed to begin. People might actually buy prime day stuff if your site wouldn't crash.
Starcom believes that Amazon Prime will be key to the company's growth in Australia, since shoppers who pay the annual Prime membership fee tend to spend more and shop more often than non-members. That generated social buzz for the product that boosted traffic when Amazon recovered, said Jeff Perkel, director of e-commerce for the San Diego-based company.
"I'm sure they are working furiously and many heads are rolling", she said.