Early in the game, before your infection gets noticed, there is a chance you will receive a message reading, "Pylons 'vital to high-tech society' A new energy report confirmed the need to construct additional pylons in order to ensure a sustainable energy supply in the future"

This is a reference to the Protoss race from Starcraft, who are required to build pylons before they can build more units.

James Vaughn, the game's developer, was invited to speak at the Center of Disease Control, or CDC for short, in March 2013. Vaughn spoke about how he modeled the spread of infection in the game as well as how Plague Inc. can raise awareness and educate the public on the spread of disease. The CDC later commented on this by saying "The game creates a compelling world that engages the public on serious public health topics".

In 2016, one of the most popular names used by players for their virus was 'Trump', according to the game's developer, Ndemic Creations. This is a reference to presidential candidate Donald Trump.

In 2019, amid a rise in popularity for anti-vaccine conspiracy theories and psuedoscience, a petition was started for Plague Inc. to implement anti-vaccine advocates, or "anti-vaxxers", as a situation that would buff your disease. Ndemic Creations, while noting that anti-vaxxers do technically exist as a smaller part of the "Science Denial" scenario along with flat Earth theorists and climate change deniers, agreed to include the movement as a solo buff for the disease, but did not have an idea for how they would work in gameplay at the time. Anti-vaxxers have not yet been added to the game, but a second buff partially referencing anti-vaxxers, "Fake News", would be added.