Background: Online social media, such as the microblogging site Twitter, have become a space for speedy exchange of information regarding sexually transmitted diseases (STDs), presenting a potential risk environment for how STDs are portrayed. Examining the types of "tweeters" (users who post messages on Twitter) and the nature of "tweet" messages is important for identifying how information related to STDs is posted in online social media. Objective: The intent of the study was to describe the types of message emitters on Twitter in relation to two different STDs-chlamydia and human immunodeficiency virus (HIV)-as well as the nature of content tweeted, including how seriously the topic was treated. Methods: We used the Twitter search engine to look for tweets posted worldwide from August 1-7, 2013, and from September 1-7, 2013, containing the words "chlamydia" or "HIV", and the hashtags "#chlamydia" or "#HIV". Tweeters were classified by two independent reviewers according to the type of avatar of the user (human, logo, or fantasy), the identification of the emitter (identifiable, semi-identifiable, or non-identifiable), and the source (private company, general media, scientific media, non-governmental, individual account, academic institution, government department, or undefined). Tweet messages were also independently classified according to their nature (serious or jokes/funny), and whether their main message was factual or of a personal nature/experience. Results: A total of 694 tweets were posted by 426 different users during the first 7 days of August and September, containing the hashtags and/or simple words "chlamydia" and/or "HIV". Jokes or funny tweets were more frequently posted by individual users (89%, 66/74), with a human avatar (81%, 60/74), from a non-identifiable user (72%, 53/74), and they were most frequently related to chlamydia (76%, 56/74). Serious tweets were most frequently posted by the general media (20.6%, 128/620), using a logo avatar (66.9%, 415/620), and with identifiable accounts (85.2%, 528/620). No government departments, non-governmental organizations, scientific media, or academic institutions posted a joke on STDs. A total of 104 of these analyzed tweets were re-tweeted messages, belonging to 68 unique tweets. The content was serious (99%, 67/68), factual (90%, 52/58), and about HIV (85%, 58/68). Conclusions: Social media such as Twitter may be an important source of information regarding STDs provided that the topic is presented appropriately. Reassuringly, the study showed that almost 9/10 of tweets on STDs (chlamydia and HIV) were of serious content, and many of the tweets that were re-tweeted were facts. The jokes that were tweeted were mainly about chlamydia, and posted by non-identifiable emitters. We believe social media should be used to an even larger extent to disseminate correct information about STDs.