I have an C++ game which sends a Python-SocketIO request to a server, which loads the requested JSON data into memory for reference, and then sends portions of it to the client as necessary. Most of the previous answers here detail that the server has to repeatedly search the database, when in this case, all of the data is stored in memory after the first time, and is released after the client disconnects.
I don't want to have a large influx of memory usage whenever a new client joins, however most of what I have seen points away from using small files (50-100kB absolute maximum), and instead use large files, which would cause the large memory usage I'm trying to avoid.
My question is this: would it still be beneficial to use one large file, or should I use the smaller files; both from an organization standpoint and from a performance one?
Is it better to have one large file or many smaller files for data storage?

Both can potentially be better. Each have their advantage and disadvantage. Which is better depends on the details of the use case. It's quite possible that best way may be something in between such as a few medium sized files.
Regarding performance, the most accurate way to verify what is best is to try out each of them and measure.
Is it better to have one large file or many smaller files for data storage?