The video-sharing app is on a list of "very large" online platforms that must comply with stricter rules under the bloc's new Digital Services Act (DSA).
Firms on the list must pay fees to the European Commission, the EU's executive arm, to finance the DSA's enforcement.
But TikTok said it opposed the way the fee had been calculated, although it confirmed it had paid the money due last year.
"We disagree with the fee and are appealing on a number of grounds, including the use of flawed third party estimates of our monthly active user numbers as a basis for calculating the total amount," a TikTok spokesperson said in a statement.
TikTok last year said it had on average 125 million monthly active users in the EU.
The milestone DSA law compels the platforms to do more to police content online and also demands they take greater action to protect online shoppers from fraud.
The commission said the companies on its list of "very large" platforms had all paid the fees due by the end of last year.
A commission spokesperson said the fee is calculated "in proportion to the size of the service" -- reflected by the number of EU users -- and does not exceed "an overall ceiling (set at 0.05 percent of the annual worldwide net income) for each provider".
Meta on Wednesday said it disagreed with the methodology used to calculate the fees.
The DSA came into force for the biggest companies last year and will apply to all firms from February 17, 2024.
The EU also faces legal challenges by online retailers Amazon and Zalando, who disagree with their designation as "very large".
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